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Recombinant DNA Technology - Dr Magrann

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					BIOTECHNOLOGY




                1
   On November 22, 1983, the sleepy
    English village of Narborough awoke to
    news of a horrific crime: A 15-year-old-
    girl named Lynda Mann had been raped
    and murdered on a country lane near her
    home. The killer left behind few clues,
    except for semen on the victim’s body and
    clothes. Despite extensive investigation,
    the trail of evidence ran cold and the
    crime went unsolved.



                                                2
   Three years later, the horror resurfaced when
    another 15-year old girl, Dawn Ashworth, was
    also raped and murdered less than a mile away
    from the first crime scene. When tests indicated
    that the 1983 and 1986 semen samples could be
    from the same man, police began to search for a
    double murderer. After another extensive
    investigation, a maintenance worker from a
    nearby hospital was arrested and charged with
    both crimes. Under considerable pressure from
    police, the worker confessed to the second
    murder, but denied committing the first.



                                                       3
DNA Technology
   In an attempt to pin both murders on the suspect, investigators
    turned to Alec Jeffreys, a professor at nearby Leicester University,
    who had recently developed the first DNA fingerprint identification
    system. Because the DNA sequence of every person unique
    (except for identical twins), DNA fingerprinting can be used to
    determine with near certainty whether two samples of genetic
    material are from the same individual. Jeffreys compared DNA
    from the 1983 and 1986 semen samples. As the police suspected,
    the DNA analysis proved that the same person had committed
    both crimes. However, when Jeffreys analyzed the suspect’s DNA,
    it did not match either crime scene sample, proving that the
    suspect must be innocent. The police quickly released the
    suspect, making him the first person in legal history to be
    exonerated by DNA evidence.




                                                                           5
   The detectives were back at square one. In an attempt to
    collect more evidence, they asked every young male from
    the surrounding area to donate blood for DNA testing.
    Although 5,000 men were sampled, none had DNA that
    matched the evidence from the crime scenes. The police
    were once again stymied. The case finally broke when a
    pub-goer described how a man named Cohn Pitchfork had
    bullied him into submitting blood on Pitchfork’s behalf. The
    police promptly ar-rested Pitchfork and took a sample of
    his blood. Indeed, his DNA matched the samples from the
    two crime scenes. Cohn Pitchfork pleaded guilty to both
    crimes, closing the first murder case ever to be solved by
    DNA evidence.




                                                                   6
   The Narborough murders were the first of many criminal
    in-vestigations that have relied on DNA evidence. DNA
    technology— methods for studying and manipulating genetic
    material—have rapidly revolutionized the field of forensics, the
    scientific analy-sis of evidence for legal investigations. Since its
    introduction, DNA fingerprinting has become a standard law
    enforcement tool and has provided crucial evidence (of both
    innocence and guilt) in many famous cases, including the O.J.
    Simpson mur-der trial and the impeachment of President Bill
    Clinton. As we will see, DNA technology has applications in many
    other fields, from cancer research to agriculture and even history.
    Perhaps the most exciting use of DNA technology in basic
    research is the Human Genome Project, whose goal was to map
    the entire human DNA down to the level of its nucleotide
    sequences. This project is expected to help us better understand
    and treat many diseases.




                                                                           7
   There are several significant roles that
    DNA technology has assumed in society,
    including gene cloning to produce useful
    products, DNA fingerprinting and forensic
    sci-ence, human gene therapy for the
    treatment of disease, compar-isons of
    genomes from different organisms, and
    the agricultural production of genetically
    modified organisms. There are also some
    of the social, legal, and ethical issues that
    are raised by these new technologies.


                                                    8
   The use of technology to alter the genomes of
    viruses, bacteria, and other cells for medical or
    industrial purposes is called genetic engineering.
   These days, bacteria, plants, and animals are genetically
    engi-neered to produce biotechnology products.
   Organisms that have had a foreign gene inserted
    into them are called trans-genic organisms.
    (TRANSferred GENE = TRANS GENIC).
   DNA technology is changing the pharmaceutical
    industry and medicine
   DNA technology, and gene cloning in particular, is widely
    used to produce medicines and to diagnose diseases.



Biotechnology Products
                                                                9
DNA technology is changing the
pharmaceutical industry and
medicine
   Therapeutic Hormones Consider human insulin
    and human growth hormone (HGH).
   In the United States alone, about 2 million people
    with diabetes depend on insulin treatment.
   Before 1982, the main sources of this hormone
    were pig and cattle tissues obtained from
    slaughterhouses.
   Insulin extracted from these animals is
    chemically similar, but not identical, to human
    insulin, and it causes harmful side effects in
    some people.
DNA technology is changing the
pharmaceutical industry and
medicine
   Genetic engineering has largely solved this
    problem by developing bacteria that synthesize
    and secrete actual human insulin.
   In 1982, because growth hormones from other
    animals are not effective in hu-mans HGH was
    urgently needed. In 1985, molecular biologists
    were able to produce HGH in bacteria.
   Before this genetically engineered hormone
    became available, children with a HGH deficiency
    had to rely on scarce supplies from human
    cadavers or else face dwarfism.
Human insulin produced by bacteria
  ◦ In 1982, Humulin became the first
    recombinant drug approved by the Food and
    Drug Administration




                  Figure 12.7A
 Recombinant DNA technology means to
  recombine the DNA of an organism to
  make it more useful to humans.
 It is used to produce bacteria that
  reproduce in large vats to get them to
  make a large amount of a particular
  protein, such as insulin, growth
  hor-mone, clotting proteins for
  hemophiliacs, and hepatitis B vaccine.



From Bacteria
                                           14
Hepatitis B Vaccine
 DNA technology is also helping medical
  researchers develop vaccines.
 A vaccine is a harmless variant or derivative of a
  pathogen (usually a bacterium or virus) that is
  used to prevent an infectious disease.
 When a person is inoculated, the vaccine
  stimulates the immune system to develop lasting
  defenses against the pathogen.
 For the many viral diseases for which there is no
  effective drug treatment, prevention by
  vaccination is virtually the only medical way to
  prevent illness.


Vaccines
 One DNA technology vaccine is for the
  hepatitis B virus.
 Hepatitis is a disabling and sometimes
  fatal liver disease, and the hepatitis B
  virus may also cause liver cancer.




Vaccines
 Smallpox was once a dreaded human
  disease, but it was eradicated worldwide
  in the 1970s by widespread vaccination
  with a harmless variant of the smallpox
  virus.
 In fact, the harmless virus could be
  engineered to carry the genes needed to
  vaccinate against several diseases
  simultaneously.
 In the future, one inoculation may prevent
  a dozen diseases.
Vaccines
Biotechnology Products From
Bacteria
   Transgenic bacteria
    can also help
    plants. For
    example, bacteria
    that live in plants
    have genes spliced
    in that let them
    resist insect
    toxins; this
    protects the roots
    of the plants, too.
   Bacteria can be
    genetically engineered
                             Biotechnology
    to degrade a             Products From
    particular substance,
    for instance,
                             Bacteria
    transgenic bacteria
    have been produced
    which have the
    ability to eat oil
    after an oil spill.
   Industry has found that bacteria can be used as
    filters to prevent airborne chemicals from being
    vented into the air.
   They can also remove sulfur from coal before it is
    burned and help clean up toxic dumps.
   Furthermore, these bacteria were given
    “suicide” genes that cause them to self-
    destruct when the job is accomplished.




From Bacteria                                        21
Biotechnology Products From
Bacteria
 Many major mining
  companies already
  use bacteria obtain
  various metals.
 Genetic
  engineering may
  enhance ability of
  bacteria to extract
  copper, uranium,
  and gold.
Biotechnology Products From
Plants

   Plants can also be
    genetically
    engineered to
    make cotton, corn,
    soybeans, and
    potatoes resistant
    to pests because
    their cells now
    produce an insect
    toxin.
Biotechnology Products From
Plants
   Plants are also being
    engineered to produce
    human hormones,
    clotting factors, and
    antibodies, in their seeds.
   One type of antibody made
    by corn can deliver a
    substance that kills tumor
    cells, and another made by
    soybeans can be used as
    treatment for genital
    herpes.
Genetically modified organisms
are transforming agriculture
 Scientists concerned with feeding the
  growing human population are using DNA
  technology to make genetically modified
  (GM) organisms for use in agriculture.
 A GM organism (Or GMO) is one that has
  acquired one or more genes by artificial
  means rather than by traditional breeding
  methods. (The new gene may or may not
  be from another species.)
Genetically modified organisms
are transforming agriculture
 To make genetically modified plants,
  researchers can manipulate the DNA of a
  single cell and then grow a plant with a
  new trait from the engineered cell.
 Already in commercial use are a number
  of crop plants carrying new genes for
  desirable traits, such as delayed ripening
  and resistance to spoilage and disease.
Genetically modified organisms
are transforming agriculture
   The most common vector used to introduce new genes into
    plant cells is a piece of DNA from a soil bacterium.
   With the help of a special enzyme, the gene for the desired
    trait is inserted into a plant cell, where it is integrated into
    a plant chromosome.
   Finally, the re-combinant cell is cultured and grows into a
    whole plant.
   If the newly acquired gene is from another species, the
    recombinant organism is called a transgenic organism.
Genetically modified organisms
are transforming agriculture
   Genetic engineering is rapidly replacing
    traditional plant-breeding programs.
   For example, the majority of the American
    soybean and cotton crops are genetically
    modified.
   Many of these GM plants have received bacterial
    genes that make the plants resistant to
    herbicides or pests.
   Farmers can more easily grow these crops with
    far less tillage and reduced use of chemical
    insecticides.
Genetically modified organisms
are transforming agriculture
   Genetic engineering also has great potential for
    improving the nutritional value of crop plants.
   “Golden rice,” a transgenic variety with a few
    daffodil genes, produces grains containing beta-
    carotene, which our body uses to make vitamin
    A.
   This rice could help prevent vitamin A
    deficiency—and resulting blindness—among the
    half of the world’s people who depend on rice as
    their staple food.
   Agricultural researchers are also making
    transgenic animals. To do this, scientists first
    remove egg cells from a female and fertilize
    them in vitro. They then inject a previously
    cloned gene directly into the nuclei of the
    fertilized eggs. Some of the cells integrate the
    foreign DNA into their genomes. The engineered
    embryos are then surgically implanted in a
    surrogate mother. If an embryo develops
    successfully, the result is a transgenic animal,
    containing a gene from a third “parent” that may
    even be of another species.

Biotechnology Products From
Animals
 Techniques have been developed to insert
  genes into the eggs of animals.
 The procedure has been used to produce
  larger fish, cows, pigs, rabbits, and sheep.
 Genetically engineered fishes are now
  being kept in ponds that offer no escape
  to the wild because there is much concern
  that they will upset or destroy natural
  ecosystems.



From Animals
                                             32
Transgenic Pig
   The goals of creating a transgenic animal
    are often the same as the goals of
    traditional breeding—for instance, to
    make a sheep with better quality wool or
    a cow that will ma-ture in a shorter time.
    Scientists might, for example, identify and
    clone a gene that causes the development
    of larger mus-cles (muscles make up
    most of the meat we eat) in one vari-ety
    of cattle and transfer it to other cattle or
    even to sheep.
Biotechnology Products From
Animals
   Transgenic animals also have been
    engineered to be phar-maceutical
    “factories” that produce otherwise rare
    biological substance for medical use.
    Recently, researchers have engineered
    transgenic chickens that express large
    amounts of the foreign product in their
    eggs. This success suggests that
    transgenic chickens may emerge as
    relatively inexpensive pharmaceutical
    facto-ries in the near future.
Biotechnology Products From
Animals
 Gene pharming is the use of
  transgenic farm animals to produce
  therapeutic drugs in the animal’s
  milk.
 There are plans to produce drugs for the
  treatment of cystic fibrosis, cancer, blood
  diseases, and other disorders.
 An anti-clotting medicine is currently
  being produced by a herd of goats.



From Animals
                                                36
Pharm Animals
 Animals have been engineered to produce
  growth hormone in their urine instead of
  in milk.
 Urine is preferable to milk because only
  females produce milk, and not until
  maturity, but all animals produce urine
  from birth.




From Animals
                                             38
39
   Scientists have begun the process of genetically
    engineering animals to serve as organ
    donors for humans who need a transplant.
   We now have the ability to transplant kidneys,
    heart, liver, pancreas, lung, and other organs.
   Unfortunately, however, there are not enough
    human donors to go round.

Fifty thousand Americans need
transplants a year, but only
20,000 patients get them.
As many as 4,000 die each year
while waiting for an organ.



Xenotransplantation                                40
   You might think that apes, such as the
    chimpanzee or the baboon might be a
    scientifically suitable species for this purpose.
   But apes are slow breeders and many people
    object to using apes for this purpose.
   In contrast, pigs have been an acceptable meat
    source, and a female pig can become pregnant
    at six months and can have two litters a year,
    each averaging about ten offspring.
   Ordinarily, the human body rejects transplanted
    pig organs. Genetic engineering, however, can
    make pig organs good for transplantation at
    less of a rejection risk.

Xenotransplantation                                     41
 Imagine that an animal has been
  genetically altered to serve as an organ
  donor. What would be the best possible
  way to get identi-cal copies of this
  animal?
 If cloning of the animal was possible, you
  could get many exact copies of this
  animal.
 Cloning is a form of asexual
  re-production (without sex) because
  it requires only the genes of that one
  animal.
Cloning of Animals
                                               42
Cloning
 In 1997, scientists at the Raslin institute
  in Scotland announced that they produced
  a cloned sheep called Dolly. In 1998,
  genetically altered calves were cloned in
  the United States using the same method.
 CLONING VIDEO




Cloning of Animals
                                                44
Clones
Clones
Clone?
 As soon as scientists realized the power of
  DNA technology, they began to worry
  about potential dangers.
 Early concerns focused on the possibility
  that recombinant DNA technology might
  create new pathogens.
 What might happen, for instance, it
  cancer cell genes were transferred into
  bacteria or viruses?

Could GM organisms harm human
health or the environment?
   To guard against such rogue microbes, scientists developed
    a set of guidelines that were adopted as formal government
    regulations in the United States and some other countries.
   One safety measure is a set of strict laboratory procedures
    designed to protect researchers from infection by
    engineered microbes and to prevent the microbes from
    accidentally leaving the laboratory.
   In addition, strains of microorganisms to be used in
    recombinant DNA experiments are genetically crippled to
    ensure that they cannot survive outside the laboratory.
   Finally, certain obviously dangerous experiments have been
    banned.



Could GM organisms harm human
health or the environment?
 Today, most public concern about possible
  hazards centers not on recombinant
  microbes but on genetically modified (GM)
  crop plants.
 Advocates of a cautious approach fear
  that some crops carrying genes from
  other species might cause allergies in
  humans or create super-weeds that are
  hazardous to the environment.

Could GM organisms harm human
health or the environment?
   Today, governments and regulatory agencies
    throughout the world are grappling with how to
    facilitate the use of biotechnology in agriculture,
    industry, and medicine while ensuring that new
    products and procedures are safe.
   In the United States, all projects are evaluated
    for potential risks by regulatory agencies such as
    the Food and Drug Administration, Environmental
    Protection Agency, National Institutes of Health,
    and Department of Agriculture.
   These agencies are under increasing pressure
    from some consumer groups.

Could GM organisms harm human
health or the environment?
   The Human Genome Project was a massive
    effort to figure out what the sequence is of
    all of the genes in human chromosomes.
    This was just finished in 2003.
   Project goals were to identify all the 25,000
    genes in human DNA and determine the
    sequences of the 3 billion amino acids that make
    up human DNA.
   This allows scientists to detect some defective
    genes and tailor a treatment plan to the
    individual.



The Human Genome Project
                                                       52
The Human Genome Project
 Gene therapy gives a patient a
  normal gene to make up for a faulty
  gene.
 For example, there is a genetic disease of
  the liver that causes it to malfunction and
  leads to high levels of blood cholesterol,
  which makes the patient subject to fatal
  heart attacks at a young age.
 The person is injected with a virus that
  contains the normal gene.

Gene Therapy
                                                54
 Another example is when fat enzymes are
  coated with the missing gene to cure
  cystic fibrosis and then sprayed into
  patients’ nostrils.
 Anti-cancer genes can also be injected
  directly into cancerous tumors.
 Perhaps it will be possible also to use
  gene therapy to cure hemophilia,
  diabetes, Parkinson disease, or AIDS.



Gene Therapy
                                            55
 DNA technology is being used increasingly
  in disease diagnosis.
 It is used to determine which genes are
  associated with genetic diseases.
 An individual’s gene expression profile
  may someday allow physicians to tailor
  treatments for many different disorders.




Diagnosis and Treatment of
Disease
   Recombinant DNA technology
    ◦ Intentionally modifying genomes of organisms
      for practical purposes
    ◦ Three goals
      Eliminate undesirable phenotypic traits
      Combine beneficial traits of two or more
       organisms
      Create organisms that synthesize products
       humans need


The Role of Recombinant DNA
technology in Biotechnology
                                                     57
Overview of
recombinant DNA
technology




                               58

                  Figure 8.1
   Mutagens
    ◦ Physical and chemical agents that produce
      mutations
    ◦ Scientists utilize mutagens to
       ◦ Create changes in microbes’ genomes to change
         phenotypes
       ◦ Select for and culture cells with beneficial characteristics
    ◦ Mutated genes alone can be isolated


The Tools of Recombinant DNA
Technology

                                                                        59
Natural Mutation in Fruit Fly
   The Use of Reverse Transcriptase to
    Synthesize cDNA
    ◦ Isolated from retroviruses
    ◦ Uses RNA template to transcribe molecule of
      DNA
    ◦ Easier to isolate mRNA molecule for desired
      protein first
      Allows cloning in prokaryotic cells



The Tools of Recombinant DNA
Technology
                                                    61
   Synthetic Nucleic Acids
    ◦ Molecules of DNA and RNA produced in cell-free
      solutions
    ◦ Uses of synthetic nucleic acids
      Elucidating the genetic code
      Creating genes for specific proteins
      Synthesizing DNA and RNA probes to locate
       specific sequences of nucleotides
      Synthesizing antisense nucleic acid molecules


The Tools of Recombinant DNA
Technology
                                                       62
   Restriction Enzymes
    ◦ Bacterial enzymes that cut DNA molecules only
      at restriction sites
    ◦ One enzyme might only cleave T-T apart;
      another enzyme only cleaves A-T apart, etc.
    ◦ We use one enzyme, and see how many pieces
      result. Then we use the next enzyme, etc. That
      shows us the sequence of DNA in a sample.
    ◦ Restriction Enzymes video

The Tools of Recombinant DNA
Technology
                                                       63
Actions of
restriction
enzymes




                           64

              Figure 8.2
   Vectors
    ◦ Nucleic acid molecules that deliver a gene into
      a cell
    ◦ Useful properties
      Small enough to manipulate in a lab
      Survive inside cells
      Contain recognizable genetic marker
      Ensure genetic expression of gene
    ◦ Include viral genomes, transposons, and
      plasmids


The Tools of Recombinant DNA
Technology
                                                        66
   Gene Libraries
    ◦ A collection of bacterial or phage clones
      Each clone in library often contains one gene of an
       organism’s genome
    ◦ Library may contain all genes of a single
      chromosome
    ◦ Library may contain set of DNA complementary
      to mRNA




The Tools of Recombinant DNA
Technology
                                                             67
 Multiplying DNA in vitro: The
  Polymerase Chain Reaction (PCR)
 VIDEO CLIP
    ◦ Large number of identical molecules of DNA
      produced in vitro
    ◦ Critical to amplify DNA in variety of situations
      Epidemiologists use to amplify genome of
       unknown pathogen
      Amplified DNA from Bacillus anthracis spores in
       2001 to identify source of spores
Techniques of Recombinant DNA
Technology
                                                         68
   Multiplying DNA in vitro: The
    Polymerase Chain Reaction (PCR)
    ◦ Repetitive process consisting of three steps
      Denaturation
      Priming
      Extension
    ◦ Can be automated using a thermocycler



Techniques of Recombinant DNA
Technology
                                                     69
Polymerase
chain
reaction
(PCR)
                           70

             Figure 8.5a
Polymerase
chain
reaction
(PCR)
                           71

             Figure 8.5b
Techniques of Recombinant DNA
Technology
                                72
   Separating DNA Molecules: Gel
    Electrophoresis and the Southern Blot
    ◦ Gel electrophoresis
      Separates molecules based on electrical charge,
       size, and shape
      Allows scientists to isolate DNA of interest
      Negatively charged DNA drawn toward positive
       electrode
      Agarose makes up gel; acts as molecular sieve
      Smaller fragments migrate faster and farther than
       larger ones
      Determine size by comparing distance migrated to
       standards

Techniques of Recombinant DNA
Technology                                                 73
Gel electrophoresis
                                   74

                      Figure 8.6
   GEL ELECTROPHORESIS VIDEO




                                75
   Separating DNA Molecules: Gel
    Electrophoresis and the Southern Blot
    ◦ Southern blot
      DNA transferred from gel to nitrocellulose
       membrane
      Probes used to localize DNA sequence of interest
      Northern blot – used to detect RNA
    ◦ Uses of Southern blots
      Genetic “fingerprinting”
      Diagnosis of infectious disease
      Demonstrate incidence and prevalence of
       organisms that cannot be cultured

Techniques of Recombinant DNA
Technology
                                                          76
The Southern
blot
technique
                            77

               Figure 8.7
   DNA Microarrays
    ◦ MICROARRAY VIDEO
    ◦ Consist of molecules of immobilized single-
      stranded DNA
    ◦ Fluorescently labeled DNA washed over array
      will adhere only at locations where there are
      complementary DNA sequences
    ◦ Variety of scientific uses of DNA microarrays
      Monitoring gene expression
      Diagnosis of infection
      Identification of organisms in an environmental
       sample

Techniques of Recombinant DNA
Technology
                                                         78
DNA microarray




                              79

                 Figure 8.8
    Inserting DNA into Cells
     ◦ Goal of DNA technology is insertion of DNA into
       cell
     ◦ Natural methods
       Transformation
       Transduction
       Conjugation
     ◦ Artificial methods
       Electroporation
       Protoplast fusion
       Injection – gene gun and microinjection


Techniques of Recombinant DNA
Technology                                           80
   Transformation is the genetic alteration of
    a cell resulting from the direct uptake,
    incorporation and expression of
    exogenous DNA from its surroundings.
    Transformation occurs naturally in some
    species of bacteria, but it can also be
    caused artificially.




Transformation
                                                  81
 Transduction is when DNA is transferred
  from one bacterium to another by a virus
 The virus is called a bacteriophage.


   Conjugation is when a bacterium uses its
    sex pilus to transfer some of its DNA to
    another bacterium.




Transduction
                                               82
Artificial methods of inserting DNA into
cells
                                                83

                                    Figure 8.9a/b
Artificial methods of inserting DNA into
cells
                                                84

                                    Figure 8.9c/d
   Genetic Mapping
    ◦ Locating genes on a nucleic acid molecule
    ◦ Provides useful facts concerning metabolism,
      growth characteristics, and relatedness to
      others
   Locating Genes
    ◦ Until 1970, genes identified by labor-intensive
      methods
    ◦ Simpler and universal methods now available
    ◦ Restriction fragmentation
    ◦ Fluorescent in situ hybridization (FISH)

Applications of Recombinant DNA
Technology
                                                        85
Fluorescent in situ hybridization


                            Used to detect and
                            localize the
                            presence or
                            absence of specific
                            DNA sequences on
                            chromosomes.

                            Also allows for
                            more precise DNA
                            karyotyping.




                                                       86

                                         Figure 8.10
Automated DNA sequencing
                                         87

                           Figure 8.11
   Environmental Studies
    ◦ Most microorganisms have never been grown in
      a laboratory
    ◦ Scientists know them only by their DNA
      fingerprints
      Allowed identification of over 500 species of
       bacteria from human mouths
      Determined that methane-producing archaea are
       a problem in rice agriculture


Applications of Recombinant DNA
Technology
                                                       88
   Pharmaceutical and Therapeutic
    Applications
    ◦ Protein synthesis
      Creation of synthetic peptides for cloning
    ◦ Vaccines
      Production of safer vaccines
      Introduce genes of pathogens into common fruits
       and vegetables
      Injecting humans with plasmid carrying gene
       from pathogen
        ◦ Humans synthesize pathogen’s proteins


Applications of Recombinant DNA
Technology                                               89
   Pharmaceutical and Therapeutic
    Applications
    ◦ Genetic screening
      DNA microarrays used to screen individuals for
       inherited disease caused by mutations
      Can also identify pathogen’s DNA in blood or
       tissues
    ◦ DNA fingerprinting
      Identifying individuals or organisms by their
       unique DNA sequence

Applications of Recombinant DNA
Technology
                                                        90
   Pharmaceutical and Therapeutic
    Applications
    ◦ Gene therapy
      Missing or defective genes replaced with normal
       copies
      Some patients’ immune systems react negatively
    ◦ Medical diagnosis
      Patient specimens can be examined for presence
       of gene sequences unique to certain pathogens
    ◦ Xenotransplants
      Animal cells, tissues, or organs introduced into
       human body


Applications of Recombinant DNA
Technology                                                91
DNA FINGERPRINT
VIDEO




DNA
fingerprinting



                                92

                  Figure 8.12
   DNA technology plays an important role in forensic
    science, the scien-tific analysis of evidence for crime
    scene and other legal in-vestigations. In violent crimes,
    body fluids or small pieces of tissue may be left at the
    crime scene or on the clothes of the victim or assailant; if
    rape has occurred, semen may be recov-ered from the
    victim’s body. With enough tissue or semen, forensic
    scientists can determine the blood type or tissue type using
    older methods that test for proteins. However, such tests
    require fresh samples in relative large amounts. Also,
    be-cause many people have the same blood or tissue type,
    this approach can only exclude a suspect; it cannot provide
    strong evidence of guilt.


DNA technology is used in courts
of law
                                                               93
   DNA testing, on the other hand, can identify the guilty
    individual with a high degree of certainty because the DNA
    sequence of every person is unique (except for identical
    twins).
   DNA testing requires only about 1,000 cells. In a murder
    case, for example, such analysis can be used to compare
    DNA samples from the suspect, the victim, and bloodstains
    on the suspect’s clothes.
   They provide a DNA fingerprint, or specific pattern of
    bands.




DNA technology is used in courts
of law
                                                                 94
   DNA fingerprinting can also be used to establish family
    relationships.
   A comparison of the DNA of a mother, her child, and the
    purported father can conclusively settle a question of
    paternity.
   Sometimes paternity is of historical interest: DNA
    fingerprinting provided strong evidence that Thomas
    Jeffer-son or one of his close male relatives fathered at
    least one child with his slave Sally Hemings.




DNA technology is used in courts
of law
                                                                95
   Just how reliable is DNA fingerprinting? In most legal
    cases, the probability of two people having identical DNA
    fingerprints is between one chance in 100,000 and one in a
    billion.
   For this reason, DNA fingerprints are now accepted as
    compelling evidence by legal experts and scientists alike.
   In fact, DNA analysis on stored forensic samples has
    provided the evidence needed to solve many “cold cases”
    in recent years.
   DNA fingerprinting has also exonerated many wrongly
    convicted people, some of whom were on death row.




DNA technology is used in courts
of law
                                                                 96
   Traditional fingerprinting has been used
    for years to identify criminals and to
    exonerate those wrongly accused of
    crimes. The opportunity now arises to
    use DNA fingerprinting in the same way.
    DNA fingerprinting requires only a small
    DNA sample. This sample and calm from
    blood left at the scene of a crime, semen
    from a rape case, even a single hair root!

DNA Fingerprinting and the
Criminal Justice System
                                                 97
   The DNA is amplified, cut with restriction
    enzymes, and separated by gel
    electrophoresis to produce a unique DNA
    fragment pattern. The same procedure is
    done several times with restriction
    enzymes, making it nearly impossible for
    anyone else in the world would have the
    same set of patterns. Advocates of DNA
    fingerprinting claim that identification is
    beyond a reasonable doubt.
DNA Fingerprinting and the
Criminal Justice System
                                                  98
   Opponents of this technology, however, point out that it is
    not without its problems. Police or laboratory negligence
    can invalidate the evidence. For example, during the O.J.
    Simpson trial, the defense claimed that the DNA evidence
    was inadmissible because it could not be proven that the
    police had not planted over Jay's blood at the crime scene.
    There have also been reported problems with sloppy
    laboratory procedures and the credibility of forensic
    experts. In addition to identifying criminals, DNA
    fingerprinting can be used to establish paternity and
    maternity, determine nationality for immigration purposes,
    and identify victims of a national disaster, such as the
    terrorist attack of September 11, 2001.


DNA Fingerprinting and the
Criminal Justice System
                                                                  99
 Considering the usefulness of DNA fingerprints, perhaps everyone
  should be required to contribute blood to create a national DNA
  fingerprint data bank. Some say however this would constitute
  an unreasonable search, which is unconstitutional.
 Would you be willing to provide your DNA for a national DNA data
  bank? What types of privacy restrictions would you want on your
  DNA?
 If not everyone, do you think that convicted felons at least should
  be required to provide DNA for a databank?
 Should all defendants have access to DNA fingerprinting (at
  government expense) to prove that they did not do a crime?
  Should this include those already convicted of crimes who want to
  reopen their cases using new DNA evidence?




DNA Fingerprinting and the
Criminal Justice System
                                                                    100
     DNA Fingerprinting and the
      Criminal Justice System
                      Blood from
Defendant’s       defendant’s clothes   Victim’s
   blood                                 blood




              Figure 12.12A                        Figure 12.12B
   Agricultural Applications
    ◦ Production of transgenic organisms
      Recombinant plants and animals altered by addition
       of genes from other organisms




Applications of Recombinant DNA
Technology
                                                       102
   Agricultural Applications
    ◦ Herbicide resistance
      Gene from Salmonella conveys resistance to
       glyphosate (Roundup)
       ◦ Farmers can kill weeds without killing crops
    ◦ Salt tolerance
      Scientists have removed gene for salt tolerance
       and inserted into tomato and canola plants
      Transgenic plants survive, produce fruit, and
       remove salt from soil


Applications of Recombinant DNA
Technology
                                                         103
   Agricultural Applications
    ◦ Freeze resistance
      Crops sprayed with genetically modified bacteria
       can tolerate mild freezes
    ◦ Pest resistance
      Bt toxin
       ◦ Naturally occurring toxin only harmful to insects
       ◦ Organic farmers used to reduce insect damage to crops
      Gene for Bt toxin inserted into various crop plants
      Genes for Phytophthora resistance inserted into
       potato crops

Applications of Recombinant DNA
Technology
                                                                 104
   Agricultural Applications
    ◦ Improvements in nutritional value and yield
      Tomatoes allowed to ripen on vine and shelf life
       increased
        ◦ Gene for enzyme that breaks down pectin suppressed
      BGH allows cattle to gain weight more rapidly,
        ◦ Have meat with lower fat content and produce 10%
        more milk
      Gene for β-carotene (vitamin A precursor)
       inserted into rice
      Scientists considering transplanting genes coding
       for entire metabolic pathways
Applications of Recombinant DNA
Technology
                                                               105
   Supremacist view – humans are of greater value
    than animals
   Long-term effects of transgenic manipulations are
    unknown
   Unforeseen problems arise from every new
    technology and procedure
   Natural genetic transfer could deliver genes from
    transgenic plants and animals into other
    organisms
   Transgenic organisms could trigger allergies or
    cause harmless organisms to become pathogenic


The Ethics and Safety of Recombinant
DNA Technology                                     106
 Studies have not shown any risks to
  human health or environment
 Standards imposed on labs involved in
  recombinant DNA technology
 Can create biological weapons using same
  technology



The Ethics and Safety of Recombinant
DNA Technology
                                             107
   Ethical Issues
    ◦   Routine screenings?
    ◦   Who should pay?
    ◦   Genetic privacy rights?
    ◦   Profits from genetically altered organisms?
    ◦   Required genetic screening?
    ◦   Forced correction of “genetic abnormalities”?



The Ethics and Safety of Recombinant
DNA Technology
                                                        108
Are Genetically Engineered Foods
Safe?
   A series of focus groups conducted by the
    Food and Drug Administration in 2000
    showed that although most participants
    believed that genetically engineered foods
    might offer benefits, they also feared
    unknown long-term health consequences
    that might be associated with the
    technology. Some say that when it comes
    to human and environmental safety, there
    should be clear evidence of the absence of
    risks.

Are Genetically Engineered Foods
Safe?                                        110
   The discovery that a genetically engineered corn called
    Star Link had inadvertently made it into the food supply
    triggered the recall of chocolate shells, tortillas, and many
    other corn-based foodstuffs from supermarkets.
   Further, the makers of Star Link were forced to buy back
    Star Link from farmers and to compensate food producers
    at an estimated cost of several hundred million dollars.
   Star Link is a type of corn that contains a foreign gene
    taken from a common soil bacterium whose insecticidal
    properties have long been known.
   About a dozen varieties of this corn, as well as potatoes
    and one variety of tomato, have now been approved for
    human consumption.




Are Genetically Engineered Foods
Safe?                                                               111
   These strains contain a gene for an insecticidal protein.
    The makers of Star Link decided to use a gene for a related
    protein. They thought that using this molecule might slow
    down the chances of pest resistance to the genetically
    modified corn. In order to get FDA approval for use in
    foods, the makers of Star Link performed the required
    tasks. Like the other now approved strains, star Linked
    was not poisonous to rodents, and its biochemical structure
    is not similar to those of most food allergens. However, it
    resisted digestion and longer than the other genetically
    modified proteins when it was put in simulated stomach
    acid and subjected to heat. Because most food allergens
    are stable like this, star Linked was not approved for
    human consumption.



Are Genetically Engineered Foods
Safe?                                                         112
   The scientific community is now trying to devise
    more tests for allergens because it has not been
    possible to determine conclusively whether or
    not this second protein is an allergen. It is also
    unclear how resistant to digestion of protein
    must be in order to be an allergen, and it is also
    unclear what degree of sequence similarity a
    potential allergen must have two unknown
    allergy and to raise concern. Therefore, it is not
    understood yet where the thresholds are for
    sensitization to food allergens and thresholds for
    the visitation of a reaction with food allergens.


Are Genetically Engineered Foods
Safe?                                                    113
   Other scientists are concerned about the
    following potential drawbacks to planting
    this variety of genetically modified corn:
    resistance among populations of the
    target past, exchange of genetic material
    between the transgenic crop and related
    plant species, and crops impact on non-
    target species. They feel that many more
    studies are needed before it can be said
    for certain that genetically modified corn
    has no ecological drawbacks.

Are Genetically Engineered Foods
Safe?                                        114
   Despite controversies, the planting of genetically
    engineered corn increased in 2001. The USDA
    reports that US armors planted genetically
    engineered corn on 26% of all corn acres, 1%
    more than in 2000. In all, US farmers planted at
    least 72 million acres with mostly genetically
    engineered corn, soybeans, and cotton. The
    public wants all of genetically engineered foods
    to be labeled as such, but this may not be easy
    to accomplish because most corn meal is derived
    from both conventional and genetically
    engineered corn. So far, there has been no
    attempt to sort out one type of food product
    from the other.
Are Genetically Engineered Foods
Safe?                                                115
Genetic Profiling
   Now that the human genome has been sequenced,
    researchers are using various means to discover which
    sequencing differences among people might forecast the
    possibility of a future disease. No doubt, there are benefits
    to genetic profiling. For example, knowledge of your genes
    might indicate your susceptibility to various types of
    cancer. This information could be used to develop a
    prevention program, including the avoidance of
    environmental influences associated with the disease.
    Also, you would be less inclined to smoke if you knew your
    genes make it almost inevitable that smoking will give you
    lung cancer. Are there any reasons not to be in favor of
    genetic profiling?




Genetic Profiling                                               117
   People, however, worry that insurance companies and
    employers could use their genetic profile against them.
    Perhaps employers will not higher, or insurance companies
    will not ensure, those who have a propensity for particular
    diseases. The federal government and about 25 states
    have passed laws prohibiting genetic discrimination by
    health insurers, and 11 have passed laws prohibiting
    genetic discrimination by employers. The legislation states
    that genetic information cannot be released to anyone
    without the subject’s permission. Is that such legislation
    enough to ally are fears of discrimination? Might an
    employer not hire you or an insurance company not ensure
    you simply because you will not grant permission to access
    your genetic profile?




Genetic Profiling                                             118
   Say that two women are being considered for the same
    position, and each meets all the basic requirements for the
    job. The first denies access to her genetic profile, while
    the second one grants permission to look at her genetic
    profile. Thinking that the first woman might have had
    something to hide, employer hires a second one. The
    possibility that sick days may be needed by the first
    woman makes the second woman a more cost-effective
    choice. People who have genetic profiles proving they are
    likely to be healthy in the future might even use them in
    order to have an advantage over those who have profile
    showing that they are likely to develop serious illnesses in
    the future. In this way, we might create a genetic
    underclass.




Genetic Profiling                                              119
   Genetic information is sometimes
    misunderstood, particularly by laypeople.
    In the past, for example, as an effort to
    combat sickle cell disease, many people
    were screened for it. Unfortunately, those
    who were found to have the sickle trait,
    and not the actual disease, experienced
    discrimination at school, or from
    employers and insurance carriers. It is
    possible misunderstanding of the results
    enough not to do genetic profiling, or do
    the potential benefits outweigh the risks?
Genetic Profiling                            120
   On the other hand, employers may fear
    that the government might use genetic
    information one day to require them to
    provide an environment specific to every
    employee's need, in order to prevent
    future illness. Would you approve of this,
    or should individuals be required to leave
    an area or job that exposes them to an
    environmental influence that could be
    detrimental to their health?



Genetic Profiling                                121
   Some people believe that free access to genetic profiling
    data is absolutely essential to developing better
    preventative care for all. If researchers can match genetic
    profiles to the environmental conditions that bring on
    illnesses, they could come up with better prevention
    guidelines for the next generation. Should genetic profiles
    and health records become public information under these
    circumstances? It would particularly help in the study of
    complex diseases, such as cardiovascular disorders, non-
    insulin-dependent diabetes, and juvenile rheumatoid
    arthritis. Perhaps there would be some way to protect
    privacy and still make the information known?




Genetic Profiling                                                 122
   If present legislation to protect privacy is
    inadequate, what can be done to truly
    keep such information private? Should
    the information be coded in some way, so
    that only the medical profession can read
    it? Should people be responsible for
    keeping the only copy of their profiles,
    which would be coded so that even they
    cannot read it? Or, do you believe that
    anyone should have access to anyone's
    profile, for whatever reason?


                                                   123
   Should people be encouraged or even required to
    have their DNA analyzed so that they can
    develop programs to possibly prevent future
    illness?
   Should employers be encouraged or required to
    provide an environment suitable to a person's
    genetic profile? Or should the individual ovoid a
    work environment that could bring on an illness?
   How come we balance individual rights with the
    public health benefits of matching genetic
    profiles to detrimental environments?




                                                    124
 Potent chemicals derived from exotic
  animals are yielding a range of treatments.
 One creature you don't want to stumble upon it
  in a dark forest is a hungry vampire bat. The 3
  inch long, pointy eared night stalker has an anti-
  clotting substance and its saliva that allows it to
  dine on an unending flow of its victim’s blood.
  There is, however, one group of people that may
  come to see the vampire bats as lifesavers.
  They are stroke patients to desperately need
  improved clot-busting drugs that prevent brain
  damage and paralysis by restoring blood flow to
  stroke ravaged tissues.
Medicine’s Wild Kingdom Time
Magazine                                            125
   That's the idea behind a new drug that 15
    US hospitals will soon begin testing. It's a
    synthetic copy of an enzyme bats secrete
    when they salivate over freshly bitten
    gray. Stroke experts are already buzzing
    because early studies in mice suggests
    patients may be able to safely receive the
    bat spit up to nine hours after they've had
    a stroke. The only clot-busting drug now
    on the market must be given within three
    hours.


                                               126
Medicine’s Wild Kingdom
   Bats are not the only scary animals that may someday
    contribute to the world's expanding medicine cabinet.
   Scientists are studying everything from Gila monsters to
    scorpions to copperhead snakes.
   The toxins these creatures use to kill their prey or ward off
    foes hold seemingly boundless potential to treat human
    diseases ranging from diabetes to brain cancer.
   Refined through millions of years of evolution, these
    substances found in animal saliva, then um, and, in some
    internal organs homed in on targets such as nerve cells
    better than most chemical combination's scientists concoct.
    And often, they circulate in the body for hours on end.




Medicine’s Wild Kingdom Time
Magazine                                                        128
   Still, turning Mother Nature’s toxins into life-saving drugs
    can be harder than killing a Python with a pebble.
   First, researchers must isolate, analyze, and synthesize
    specific compounds in such a way that they can be
    tolerated by humans and mass produced.
   The risk of failure is so high that many pharmaceutical
    companies shun poison derived experimental drugs until
    they are well past the developmental stage.
   That leaves scientists dependent upon scarce venture
    capital and public funding.




Medicine’s Wild Kingdom Time
Magazine                                                           130
   Scientists are also racing against a biological clock.
   Species identified to date may represent just 1/10 of the
    biological diversity on earth.
   And potentially therapeutic creatures are vanishing at
    unprecedented rates.
   Although the vampire bat is not endangered, 13 other bat
    species are.
   The natural world is the largest pharmaceutical factory we
    have, and a lot of potential benefit is being lost.




Medicine’s Wild Kingdom Time
Magazine                                                         131
   Drugs like the synthesized bat spit can earn attractive
    returns.
   An anticoagulant derived from leach saliva pulled in an
    estimated $38 million last year.
   And a hypertension drug derived from the venom of the
    South American viper drew more than $1 billion in annual
    sales before the compounds became available in generic
    form in the mid-1990s.




Medicine’s Wild Kingdom Time
Magazine                                                       132
   Many animal poisons have the ability to hit specific bull’s-
    eyes in the body.
   One species of snail, for example, injects its prey with a
    poison that paralyzes nerve cells.
   The tiny Ecuadorian Poison Dart Frog secretes a skin toxin
    that keeps predators at bay.
   Both substances block pain signals to the brain and have
    led to experimental pain medications that could be as
    potent as morphine but with no risk of addiction.
   The poison in puffer fish has also been found to ease the
    pain of heroin withdrawal and cancer.




Medicine’s Wild Kingdom Time
Magazine                                                           134
   For sheer horror, nothing matches staining of the 8 inch
    giant yellow Israel scorpion.
   It packs neurotoxins that can cause excruciating pain.
   Yet at least one of the hundreds of proteins involved in that
    process also has the ability to seek out and find to a
    receptor that is abnormally expressed on the surface of
    brain tumor cells but not on normal selves.




Medicine’s Wild Kingdom Time
Magazine                                                        137
   Last year, to cancer centers began testing a copy of the
    protein that was developed from this toxin.
   The researchers compared the synthesized protein with a
    radioactive isotope and injected it into the brains of clinical
    trial patients suffering from a cancer called glioma.
   In the brain, they believe, the drug travels straight to the
    tumors and kills them without damaging nearby healthy
    cells. It's like a guided missile.




Medicine’s Wild Kingdom Time
Magazine                                                          139
   Exotic animals and their secretions don't necessarily have
    to be lethal to help humans.
   One pharmaceutical company hopes to obtain food and
    drug administration approval for a diabetes drug derived
    from a hormone that Gila monsters secrete while munching
    on mice, bird eggs, and other favorite foods.
   This substance mimics the human hormone that regulates
    insulin, which in turn controls blood sugar.
   But unlike the human molecule, which is quickly degraded
    by enzymes in the body, the lizard version sticks around for
    hours.
   And it helps the body regenerate insulin making cells. This
    can take us to new levels of blood sugar control.



Medicine’s Wild Kingdom Time
Magazine                                                       140
   Despite the promise of animal-based drugs, the path from
    the rain forest to the FDA is rough with pitfalls.
   For many companies, the biggest challenge has been
    figuring out how to develop and produce chemical copies of
    naturally occurring substances.
   A professor at the University of Southern California School
    of medicine in has been developing a cancer drug based on
    the venom of the Southern copperhead snake.
   With a shoestring budget from public grants, he managed
    to coax mammalian cells to make copies of the venom
    protein.
   But it will be at least a year before a drug is ready for
    human testing.



Medicine’s Wild Kingdom Time
Magazine                                                      141
   Emerging technologies should help speed the discovery and
    development of exotic drugs.
   Computerized screening systems, for example, allow
    researchers to test experimental compounds against
    thousands of potential disease targets simultaneously.
   That's important because science has barely scratched the
    surface of nature’s therapeutic potential.
   There are 10 million organisms out there waging chemical
    warfare against each other; the abundance of possible
    drugs can not even be imagined.




Medicine’s Wild Kingdom Time
Magazine                                                    142
   The problem is that many of these potential remedies are
    disappearing before they are even spotted.
   Half of the world's plants and animals live in tropical
    forests, and most of these species are still unknown.
   At the current rate of forest destruction, two thirds of land
    dwelling plant and animal species will be extinct by the end
    of this century.




Medicine’s Wild Kingdom Time
Magazine                                                        143
   The urgency of preserving nature's bounty is not lost on
    patients like Duane Rualo. The 24-year-old accounting
    student at Cal State Long Beach was diagnosed with
    glioma in late 2001 and told he probably would not live
    long enough to make it to his fall 2003 graduation. After
    surgery and several shots of scorpion venom, his latest
    brain scan came up clear of cancer. His doctors can't say
    yet how important the scorpion has been to his recovery.
    But these days, Rualo pauses when he comes across a
    scorpion exhibit at a zoo. “I stop and think:’ Wow, they
    may have saved my life’”, he says. And who knows what
    other life-saving drugs may be lurking beyond the
    scorpions layer, the Gila monsters burrow, and the bats
    caves?



Medicine’s Wild Kingdom Time
Magazine                                                        144
New Cures on the Horizon
   Now that we know the sequence of the
    bases in the DNA of all the human
    chromosomes, biologists all over the
    world believe this knowledge will result in
    rapid medical advances for ourselves and
    our children.




New Cures on the Horizon                          146
   First prediction: Many new medicines tailored to the
    individual will be available.
   Most drugs are proteins or small chemicals that are able to
    interact with proteins. Today's drugs were usually
    discovered in a hit or miss fashion, but now researchers
    will be able to take a more systematic approach to finding
    effective medicines. In a recent search for a menace and
    that makes wounds heal, researchers cultured skin cells
    with 14 proteins that can cause skin cells to grow. Only
    one of these proteins actually made skin cells grow and did
    nothing else. They expect this protein to become an
    effective drug for conditions such as venous ulcers, which
    are skin lesions that affect many thousands of people in
    the United States. Tests leading to effective medicines can
    be carried out with many more proteins that scientists will
    discover by examining the human genome.


New Cures on the Horizon                                      147
 Many drugs potentially have unwanted
  side effects. Why do some people and not
  others have won or more of the side
  effects?
 Most likely, this is because people have
  different genetic profiles.
 It is expected that a physician will be able
  to match patients to drugs that are safe
  or them on the basis of their genetic
  profiles.



New Cures on the Horizon                     148
 One study found that various
  combinations of mutations can lead to the
  development of asthma. A particular
  medicine, called albuterol, is effective and
  safe for patients with certain combinations
  of mutations and not others.
 This example and others showed that
  many diseases are multi-factorial and that
  only a genetic profile is able to detect
  mutations are causing an individual to
  have a disease and how it should be
  properly treated.
New Cures on the Horizon                     149
 Second prediction: A longer and healthier life will be yours.
 Pre-embryonic gene therapy may become routine once we
  discovered the genes that contribute to a longer and healthier
  lives. We know that the presence of three radicals causes cellular
  molecules to become unstable and cells to die. Certain genes are
  believed to code for antioxidant enzymes that detoxify free
  radicals.
 It could be that human beings with particular forms of these
  genes have more efficient antioxidant enzymes, and therefore
  live longer. If so, researchers will no doubt be able to locate
  these genes and also others that promote a longer, healthier life.
  Perhaps certain genetic profiles allow some people to live far
  beyond the normal lifespan.
 Researchers may be able to find which genes allow individuals to
  live a long time and to make them available to the general public.
  Then, many more people would live longer and healthier lives.




New Cures on the Horizon                                          150
   Third prediction: You will be able to design
    your children.
   Genome sequence data will be used to identify many more
    mutant genes that cause genetic disorders than are
    presently known.
   In the future, it may be possible to cure genetic disorders
    before the child is born by adding a normal gene to any
    egg that carries a mutant gene.
   Or an artificial chromosome, constructed to carry a large
    number of corrective genes, could automatically be placed
    in eggs.
   In vitro fertilization would have to be utilized in order to
    take advantage of such measures for current genetic
    disorders before conception.




New Cures on the Horizon                                           151
 Genome sequence data can also be used
  to identify genes for traits such as height,
  intelligence, or behavioral characteristics.
 A couple could decide on their own which
  genes may wish to use to enhance a
  child's phenotype.
 In other words, the sequencing of the
  human genome may bring about a
  genetically just society, in which all types
  of genes would be accessible to all
  parents.


New Cures on the Horizon                         152
   Finished!
Congratulations!




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